Abstract:SUMMARY
The canonical function of kinases is to transfer a phosphoryl group to substrates, initiating a signaling cascade; while their non-canonical role is to bind other kinases or substrates, acting as scaffolds, competitors, and signal integrators. Here, we show how to uncouple kinases dual function by tuning the binding cooperativity between nucleotide (or inhibitors) and substrate allosterically. We demonstrate this new concept for the C-subunit of protein kinase A (PKA-C). Using thermocalorimetry and NMR… Show more
“…Furthermore, R133A exhibited a nearly 200-fold higher IC 50 value for inhibition by PKI (Supplementary Figure S4), confirming loss of enzyme inhibition despite unchanged autophosphorylation and ATP affinity. Interestingly, the binding of both PKI (pseudosubstrate inhibitor) and ATP/Mg (nucleotide) to the WT C-subunit was additive, or even synergistic, over a range of ATP concentrations (Figure 4B), confirming that DSF detects co-operative binding at two separate sites on PKAc, consistent with recent work [41]. Figure 4.PKI protein binds stably to PKAc WT, but not K72H or R133A protein.( A ) Native ESI mass spectra of PKAc WT, K72H and R133A, in the absence or presence of equimolar PKI.…”
Section: Resultssupporting
confidence: 89%
“…Moreover, different C- and R-subunits can co-assemble to generate distinct quaternary complexes, although their composition and stoichiometry are potentially susceptible to artifacts of crystallization. Importantly, intrinsic co-operativity within the C-subunit exists [40], so that ligand binding is sensed conformationally, potentially coupling ATP site occupancy with substrate dynamics during the catalytic cycle [41]. Classes of small-molecule or protein ligands might therefore exhibit differential effects on PKA signaling, potentially through distinct binding modes, as noted for several kinase and inhibitor combinations [42,43].…”
cAMP-dependent protein kinase (PKA) is an archetypal biological signaling module and a model for understanding the regulation of protein kinases. In the present study, we combine biochemistry with differential scanning fluorimetry (DSF) and ion mobility–mass spectrometry (IM–MS) to evaluate effects of phosphorylation and structure on the ligand binding, dynamics and stability of components of heteromeric PKA protein complexes in vitro. We uncover dynamic, conformationally distinct populations of the PKA catalytic subunit with distinct structural stability and susceptibility to the physiological protein inhibitor PKI. Native MS of reconstituted PKA R2C2 holoenzymes reveals variable subunit stoichiometry and holoenzyme ablation by PKI binding. Finally, we find that although a ‘kinase-dead’ PKA catalytic domain cannot bind to ATP in solution, it interacts with several prominent chemical kinase inhibitors. These data demonstrate the combined power of IM–MS and DSF to probe PKA dynamics and regulation, techniques that can be employed to evaluate other protein-ligand complexes, with broad implications for cellular signaling.
“…Furthermore, R133A exhibited a nearly 200-fold higher IC 50 value for inhibition by PKI (Supplementary Figure S4), confirming loss of enzyme inhibition despite unchanged autophosphorylation and ATP affinity. Interestingly, the binding of both PKI (pseudosubstrate inhibitor) and ATP/Mg (nucleotide) to the WT C-subunit was additive, or even synergistic, over a range of ATP concentrations (Figure 4B), confirming that DSF detects co-operative binding at two separate sites on PKAc, consistent with recent work [41]. Figure 4.PKI protein binds stably to PKAc WT, but not K72H or R133A protein.( A ) Native ESI mass spectra of PKAc WT, K72H and R133A, in the absence or presence of equimolar PKI.…”
Section: Resultssupporting
confidence: 89%
“…Moreover, different C- and R-subunits can co-assemble to generate distinct quaternary complexes, although their composition and stoichiometry are potentially susceptible to artifacts of crystallization. Importantly, intrinsic co-operativity within the C-subunit exists [40], so that ligand binding is sensed conformationally, potentially coupling ATP site occupancy with substrate dynamics during the catalytic cycle [41]. Classes of small-molecule or protein ligands might therefore exhibit differential effects on PKA signaling, potentially through distinct binding modes, as noted for several kinase and inhibitor combinations [42,43].…”
cAMP-dependent protein kinase (PKA) is an archetypal biological signaling module and a model for understanding the regulation of protein kinases. In the present study, we combine biochemistry with differential scanning fluorimetry (DSF) and ion mobility–mass spectrometry (IM–MS) to evaluate effects of phosphorylation and structure on the ligand binding, dynamics and stability of components of heteromeric PKA protein complexes in vitro. We uncover dynamic, conformationally distinct populations of the PKA catalytic subunit with distinct structural stability and susceptibility to the physiological protein inhibitor PKI. Native MS of reconstituted PKA R2C2 holoenzymes reveals variable subunit stoichiometry and holoenzyme ablation by PKI binding. Finally, we find that although a ‘kinase-dead’ PKA catalytic domain cannot bind to ATP in solution, it interacts with several prominent chemical kinase inhibitors. These data demonstrate the combined power of IM–MS and DSF to probe PKA dynamics and regulation, techniques that can be employed to evaluate other protein-ligand complexes, with broad implications for cellular signaling.
“…A clear extension from this work will be to explore whether the mechanisms identified here are observed for other AGC kinases as well as non-AGC kinases. For example, in agreement with previous studies, we observed that both PKC and PKA show positive cooperativity between substrate and ATP binding (43). We posit that it is likely not a universal kinase mechanism as a recent paper (44) found that negative cooperativity is observed in the tyrosine kinases Src, Abl, and Hck.…”
Section: Journal Of Biological Chemistry 21967supporting
The overlapping network of kinase-substrate interactions provides exquisite specificity in cell signaling pathways, but also presents challenges to our ability to understand the mechanistic basis of biological processes. Efforts to dissect kinase-substrate interactions have been particularly limited by their inherently transient nature. Here, we use a library of FRET sensors to monitor these transient complexes, specifically examining weak interactions between the catalytic domain of protein kinase C␣ and 14 substrate peptides. Combining results from this assay platform with those from standard kinase activity assays yields four novel insights into the kinase-substrate interaction. First, preferential binding of non-phosphorylated versus phosphorylated substrates leads to enhanced kinase-specific activity. Second, kinase-specific activity is inversely correlated with substrate binding affinity. Third, high affinity substrates can suppress phosphorylation of their low affinity counterparts. Finally, the substrate-competitive inhibitor bisindolylmaleimide I displaces low affinity substrates more potently leading to substrate selective inhibition of kinase activity. Overall, our approach complements existing structural and biophysical approaches to provide generalizable insights into the regulation of kinase activity.The canonical role of a protein kinase is to recognize, bind, and phosphorylate specific serine, threonine, or tyrosine residues on a substrate protein (1, 2). Given that kinases are one of the largest gene families in eukaryotes and are involved in nearly every cellular function (3), significant work has focused on understanding the mechanisms that dictate substrate-kinase pairings (4). Although the catalytic domains of eukaryotic kinases are structurally conserved (5, 6), the local environment around the substrate binding pocket of the kinase catalytic domain varies between kinases (7). This has led to the view that phosphorylation site recognition occurs through conserved residues flanking the phospho-residue on the substrate. Linear sequence motifs have now been identified for most kinases through a combination of structural analysis and peptide libraries (4, 8). Additionally, these phospho-sites are frequently found in unstructured regions of the substrate protein that may allow for more flexible accommodation in the kinase active site (9, 10). After recognizing the substrate, the catalytic domain then transfers a phosphate group from ATP to the phosphoresidue on the substrate. Phospho-transfer is followed by release of ADP and the phosphorylated substrate to prime the kinase for another catalytic cycle (11).Crystallographic and NMR approaches have provided detailed structural information on the catalytic domains of individual kinases in complex with a variety of nucleotide analogs and inhibitors (6, 12, 13). However, the lack of defined secondary structure surrounding the phospho-motif and the inherent transient nature of the interaction has limited efforts to dissect the different states of the ...
“…An interesting functional aspect observed in some kinases is substrate binding cooperativity. Experimental studies of the cyclic AMP-dependent PK (PKA), considered to be a prototypical member of the kinase family, indicate that binding first the ATP increases the affinity toward the protein substrate (11)(12)(13). A puzzling fact is that a single point mutation of tyrosine 204, which does not form interactions with any of the substrates, to alanine (Y204A) decouples ligand binding to the catalytic subunit but does not alter its crystal structure (11,14).…”
Substrate binding cooperativity in protein kinase A (PKA) seems to involve allosteric coupling between the two binding sites. It received significant attention, but its molecular basis still remains not entirely clear. Based on long molecular dynamics of PKA and its complexes, we characterized an allosteric pathway that links ATP binding to the redistribution of states adopted by a protein substrate positioning segment in favor of those that warrant correct binding. We demonstrate that the cooperativity mechanism critically depends on the presence of water in two distinct, buried hydration sites. One holds just a single water molecule, which acts as a switchable hydrogen bond bridge along the allosteric pathway. The second, filled with partially disordered solvent, is essential for providing a smooth free energy landscape underlying conformational transitions of the peptide binding region. Our findings remain in agreement with experimental data, also concerning the cooperativity abolishing effect of the Y204A mutation, and indicate a plausible molecular mechanism contributing to experimentally observed binding cooperativity of the two substrates.
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